Field of the Invention
The present invention relates to methods and systems for cell lysis. More specifically, embodiments of the present invention relate to methods and systems for rapid continuous flow cell lysis in a microfluidic device.
Discussion of the Background
Cell lysis has many different applications. To access the DNA from cells (including the enriched bacterial DNA in the circulating neutrophils of patients with bloodstream infections), it is necessary to lyse the cells. This can be challenging when a large number of cells need to be processed in a short amount of time (such as when lysing the neutrophils recovered from patients with bloodstream infections) in a microfluidic channel. For this case, the lysis must be “continuous-flow,” which implies that traditional chemical lysis techniques cannot be used. In particular, the rapid processing of whole blood cells is useful for the diagnosis of bloodstream infections. While healthcare providers wait on current methods to process whole blood, it is necessary to use broad-range therapies to treat the infected patient, resulting in higher healthcare costs and increasing antibiotic resistance of pathogenic bacteria.
Although a number of techniques have reportedly been demonstrated for on-chip cell lysis, including chemical (Mun et al., Microfluid. Nonofluid. 8:695, 2009; Schilling et al., Anal. Chem. 74:1798, 2002), electrical (Church et al., Biomicrofluidics 4:044101, 2010; Wang et al., Biosens. Bioelectron. 22:582, 2006), acoustic (Belgrader et al., Anal. Chem. 71:4232, 1999; Taylor et al., Anal. Chem. 73:492, 2001), and optical (Lee et al., Lab Chip 6:886, 2006; Quinto-Su et al., Lab Chip 8:408, 2008) techniques, these methods require a high residence time in the microfluidic channel, and thus cannot be used for continuous-flow processing. Mechanical shearing of cells can offer the lysis speed to enable continuous-flow processing (e.g. 100 μL/min). The use of mechanical shearing for continuous-flow cell lysis has been reported in silicon devices using narrow channels with features on the scale of a few microns (see Di Carlo et al., Lab Chip 3:287, 2003; Wurm and Zeng, Lab Chip 12:1071, 2012). However, as the devices are silicon-based, they are not replica molded and thus are quite expensive and time-consuming to produce. Fabricating the devices with soft-lithography would dramatically improve the cost, but unfortunately soft materials are not compatible with the mechanical lysis concept due to significant channel expansion that such materials experience under the high fluid pressure required for continuous flow cell lysis.
The introduction of soft-lithography fabrication using the elastomer polydimethylsiloxane (PDMS) resulted in an expansion of research in the field of microfluidics (Duffy et al., Anal. Chem. 70:4974, 1998).
Two primary advantages of elastomer-based soft lithography drove this expansion of microfluidics research: (i) the low cost and ease of soft-lithography-based fabrication enabled rapid prototyping of devices, and (ii) the flexibility of the PDMS elastomer could be leveraged to fabricate actuated components, in particular valves and pumps (Unger et al., Science 288:113, 2000), into the microsystems. Thus, PDMS and soft lithography assisted in the evolution of microfluidic devices to a paradigm in which functional devices are fabricated through replica molding, which decreased the cost per device as compared to silicon- or glass-based devices.
While it is clear that soft lithography may be a valuable technique to the field of microfluidics, PDMS as a material for soft lithography has disadvantages in many important applications. For example, the vapor porosity of PDMS leads to evaporation of samples, the hydrophobicity of PDMS leads to challenges with wettability, and the deformability causes feature sizes to change under various pressure conditions. In particular, this lack of rigidity prohibits the use of PDMS for a number of applications that require fixed and predictable channel dimensions under high pressure.
One such application for which PDMS is inappropriate is rapid and continuous-flow cell lysis in a microfluidic channel.
Other polymers used as materials for soft lithography is off-stoichiometry thiol-ene (OSTE) polymer. OSTE polymers have been directly bonded using the reactive groups present on the surface, such as the bonding of two OSTE prepolymers, one OSTE prepolymer with an excess of thiol groups and the other OSTE prepolymer with an excess of allyl (or -ene) groups. The two prepolymers will react to covalently link themselves under UV exposure. One disadvantage of this method is that it requires two OSTE prepolymer formulations for bonding. The bonding of an OSTE prepolymer to itself has been reported (see Sikanen et al., J. Micromech. Microeng. 23:037002, 2013). In this case, two pieces of fully cured OSTE polymer made from an OSTE prepolymer with excess allyl groups were contacted and exposed to UV. Although bonding can be achieved with this method, contacting fully cured OSTE polymer pieces results in a bond strength that varies across assembled devices. In principle, full curing of the OSTE polymer will consume all (or most) of the thiol groups, leaving allyl groups at the surface. The allyl groups cannot react with each other to form covalent bonds. For the two fully cured pieces to bond, unreacted thiol groups must be present at the surface. The small number of unreacted thiol groups present after fully curing is not sufficient to achieve consistent bonding.
Although various methods exist to lyse cells, none of these methods describes a single device that is capable of rapid continuous flow cell lysis. Thus, there is a need to develop microfluidic systems and methods for rapid continuous flow cell lysis.